1. Field of the Invention
This invention relates to selective targeting of cells with cytotoxic genes using fingerprinting promoter driven specific cytotoxic genetic constructs and transcription factors, and to methods for using these constructs and transcription factors to treat diseases of the pancreas, and, in particular, to an RIP-tk (rat insulin promoter-thymidine kinase) construct that selectively targets insulin secreting cells, such as beta (xcex2) cells, and certain human pancreatic ductal carcinoma cells, to cause cell death.
2. Description of the Background
xcex2-cell adenomas (insulinomas) are the most common of the islet cell tumors. Ninety percent of xcex2-cell tumors are benign, however morbidity associated with their removal is significant. Malignant insulinomas have a 63% five-year recurrence rate with an average survival less than four years (Proye, C, 68 Aust. N.Z.J. Surg. 90-100 (1998)). Furthermore, there is no effective medical treatment for the devastating symptoms associated with hyperinsulinemia as a result of either insulinoma or nesidioblastosis (idiopathic hyperinsulinemia). Pancreatic ductal adenocarcinoma (PDA) likewise remains a devastating disease with a less than three percent five year survival rate.
Over 28,000 patients will be diagnosed with pancreatic cancer this year of which over 27,000 will die of their disease within five years (Yeo Cj, et al., Neoplasms of the Pancreas Exocrine Tumors in Sabiston Textbook of Surgery 1171-1175 (Sabiston D C, et al. eds., 1997)). The majority of pancreatic tumors arise from ductular cells, resembling cells found in the early embryonic pancreas. Currently, only surgery offers any chance for a cure and the majority of the time the cancer has spread before it is detected.
Cancer specific promoters are being identified and are being used in an effort to modify the expression of thymidine kinase in tumor cells (Tanaka T, et al., 231 Biochemical and Biophysical Research Communications 775-779 (1997); DiMaio J, et al., 116[2] Surgery 205-213 (1994); Osaki T, et al., 54 Cancer Research 5258-5261 (1994); Kaneko S, et al., 55 Cancer Research 5283-5287 (1995); Robertson M, et al., 5[5] Cancer Gene Ther. 331-336 (1998); Siders W, et al., 5[5] Cancer Gene Ther. 181-291 (1998); Vandier D, et al., 58 Cancer Research 4577-4590 (1998)). However, these therapies have limitations due to either the weakness of the promoter or the tissue specificity of its activation. The herpes simplex thymidine kinase (HSVtk) gene, under the transcriptional control of a ubiquitous promoter, has been introduced into a host and caused significant cell death in the presence of ganciclovir (Bonnekoh B, et al., 104 The Journal of Investigative Dermatology 313-317, (1995); Al-Hendy A, et al., 43 Gynecologic and Obstetric Investigation 268-275 (1997); Eastham J, et al., 7 Human Gene Therapy 515-523 (1996); Chen S-H, et al., 91 Proc. Natl. Acad. Sci. USA 3054-3057 (1994); Tong X, et al., 61 Gynecologic Oncology 175-179 (1996). Ganciclovir (GCV), an analogue of guanosine, requires both mammalian and viral tk to become active. In viral thymidine kinase containing cells, GCV is phosphorylated into an intermediate that kills dividing cells by inhibiting DNA synthesis and acting as a chain terminator (Mathews T, et al., 10 Rev Infect Dis. 180-192 (1992); Moolten F L, 50 Cancer Res. 7820-7825 (1986)). However, thymidine kinase with a ubiquitous promoter is not cell specific, limiting its use as a cytotoxic agent.
With the identification of tissue specific promoters, one can target therapies and selectively turn on genes in specific cell types, important goals in gene therapy. Cell specific strategies depend on a cell specific promoter that can activate the suicide gene only in the targeted tumor. To accomplish this, activation of the promoter-suicide construct should require the presence of transcription factors in the targeted tumor that will activate the promoter. In addition, an effective gene delivery system is needed. However, prior to the present invention, a method to express selected genes solely in xcex2 cells and other cells of pancreatic origin had not been developed. Such a method would provide a useful tool for development of treatment for insulinoma, nesidioblastosis and other pancreatic cancers.
Currently, there is no effective treatment for pancreatic xcex2-cell tumors or pancreatic ductal adenocarcinomas. Consequently, there is a need for an effective and selective treatment for these diseases, as well as other diseases due to abnormal pancreatic cells.
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides a novel recombinant nucleic acid comprising an RIP-tk construct useful for the selective targeting and ablation of cells, such as cells comprising one or more specific transcription factors. The invention is particularly useful for the treatment of pancreatic cancer and other diseases of or affecting the pancreas.
Accordingly, one embodiment of the invention is directed to a method for selectively expressing a target gene in a pancreatic cell comprising delivering to the cell an effective amount of an agent containing a recombinant nucleic acid sequence, the sequence comprising an insulin promoter, such as a rat insulin promoter, operatively linked to the target gene. The cell may naturally contain or be cotransfected with one or more insulin promoter transcription factors selected from the group consisting of BETA2, GATA4, E47 and PDX-1.
Another embodiment of the invention is directed to a method for selectively ablating pancreatic cells in an individual comprising administering to the individual an effective amount of an agent comprising a recombinant nucleic acid sequence. The recombinant nucleic acid sequence preferably comprises a cytotoxic gene operatively linked to an insulin promoter, such as the rat insulin promoter. The pancreatic cells preferably express one or more transcription factors selected from the group consisting of BETA2, GATA4, E47 and PDX-1. Preferably, the cytotoxic gene is a nucleic acid encoding viral thymidine kinase (tk), and the method further comprises the step of administering ganciclovir, acyclovir, FIAU or 6-methoxypurine arabinoside to the individual in an amount effective to ablate the cells.
Another embodiment is directed to a method for selectively ablating a target in an individual comprising transfecting the individual with an agent comprising a recombinant nucleic acid sequence comprising a thymidine kinase gene operatively linked to an insulin promoter, such as a rat insulin promoter, and administering to the individual an effective amount of ganciclovir, acyclovir, FIAU or 6-methoxypurine arabinoside in an amount sufficient to cause ablation of the target. Preferably, the target comprises cells that naturally express or are cotransfected with one or more rat insulin promoter transcription factors selected from the group consisting of BETA2, GATA4, E47 and PDX-1.
Another embodiment is directed to a method for the production of a protein in a cell comprising delivering a nucleic acid molecule to the cell, wherein the nucleic acid molecule comprises an insulin promoter operatively linked to a structural nucleic acid sequence encoding the protein, and the cell comprises the PDX-1 transcription factor after delivery of the nucleic acid molecule.
Another embodiment of the invention is directed to a method for ablating cells in an individual comprising delivering an agent to the individual, wherein the agent comprises a nucleic acid molecule, and the nucleic acid molecule comprises an insulin promoter operatively linked to a structural nucleic acid sequence encoding a cytotoxic protein.
Another embodiment of the invention is directed to a method for treating a metabolic disease, such as hypoglycemia or hyperinsulinemia, in an individual comprising administering to the individual an effective amount of an agent comprising a recombinant nucleic acid sequence comprising a cytotoxic gene operatively linked to an insulin promoter. Preferably, the cytotoxic gene encodes thymidine kinase and the method further comprises the step of administering to the individual an agent, such as ganciclovir.
Another embodiment of the invention is directed to a method of treating a metabolic disease in an individual comprising delivering an agent to the individual, wherein the agent comprises a nucleic acid molecule, and the nucleic acid molecule comprises an insulin promoter operatively linked to a structural nucleic acid sequence encoding a cytotoxic protein.
Another embodiment is directed to a composition for selectively causing regression or ablation of a pancreatic cell comprising a recombinant nucleic acid sequence which comprises a thymidine kinase gene operatively linked to an insulin promoter, such as the rat insulin promoter.
Another embodiment of the invention is directed to an isolated nucleic acid molecule comprising an insulin promoter operatively linked to a structural nucleic acid sequence encoding a cytotoxic protein.
Another embodiment of the invention is directed to a kit comprising an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule comprises an insulin promoter operatively linked to a structural nucleic acid sequence encoding a cytotoxic protein. Preferably, the nucleic acid molecule is contained in a first container, and the kit further comprises one or more agents selected from the group consisting of ganciclovir, acyclovir, FIAU, and 6-methoxypurine arabinoside contained in the same or a second container.
Another embodiment of the invention is directed to a method for increasing the secretion of insulin in an individual comprising reducing the concentration of the somatostatin receptor.
Other embodiments and advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of the invention.